DE19541844C1 - Process for the production of human antibodies and their use - Google Patents
Process for the production of human antibodies and their useInfo
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- DE19541844C1 DE19541844C1 DE19541844A DE19541844A DE19541844C1 DE 19541844 C1 DE19541844 C1 DE 19541844C1 DE 19541844 A DE19541844 A DE 19541844A DE 19541844 A DE19541844 A DE 19541844A DE 19541844 C1 DE19541844 C1 DE 19541844C1
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
Abstract
Description
Die Erfindung betrifft ein Verfahren zur Herstellung von menschlichen monoklonalen Antikörpern nach dem Oberbegriff von Patentan spruch 1 und deren Verwendung.The invention relates to a method for producing human monoclonal antibodies according to the preamble of Patentan saying 1 and their use.
Antikörper finden eine sehr breite Anwendung in der Behandlung und Therapie von Menschen. Antikörper werden verwendet in der Immunprophylaxe, bei der Behandlung akuter Infektionen sowie bei der Behandlung immunsupprimierter Patienten. Menschliche Antikörper sind heute nicht in ausreichender Menge herstell bar. Ein Verfahren der gattungsgemäßen Art ist z. B. aus Casali et al., (1986), Science 234, 476-479, bekannt.Antibodies are widely used in treatment and therapy of people. Antibodies are used in the Immunoprophylaxis, in the treatment of acute infections as well in the treatment of immunosuppressed patients. Human Antibodies cannot be produced in sufficient quantities today bar. A method of the generic type is such. B. from Casali et al., (1986) Science 234, 476-479.
Es werden deswegen häufig tierische Antikörper eingesetzt. Tierische Antikörper haben den Nachteil, daß sie beim Menschen störende Abwehrreaktionen hervorrufen und ihre Wirkung verlo ren geht. Menschliche Antikörper kann man aus menschlichem Serum gewinnen: Dieses Verfahren hat drei wesentliche Nachtei le: Geeignetes menschliches Serum steht nur in begrenztem Um fang zur Verfügung, die gereinigten Antikörper sind immer po lyklonal, die Behandlung ist sehr teuer.Animal antibodies are therefore often used. Animal antibodies have the disadvantage that they are found in humans cause disruptive defense reactions and lose their effectiveness ren goes. Human antibodies can be made from human Obtaining Serum: This procedure has three major disadvantages le: Suitable human serum is only available to a limited extent available, the purified antibodies are always po lyklonal, the treatment is very expensive.
Aufgabe der Erfindung ist es, ein Verfahren der eingangs ge nannten Art so auszugestalten, daß monoklonale, menschliche Antikörper in ausreichender Menge und hoher Spezifität herge stellt werden können.The object of the invention is to provide a method of ge named kind so that monoclonal, human Antibodies in sufficient quantity and high specificity can be put.
Diese Aufgabe wird durch die kennzeichnenden Merkmale des Pa tentanspruchs 1 gelöst. Die Unteransprüche beschreiben vor teilhafte Ausgestaltungen des Verfahrens. Die durch das erfin dungsgemäße Verfahren hergestellten Antikörper werden bevor zugt in einer pharmazeutischen Zubereitung als Therapeutika eingesetzt.This task is characterized by the characteristic features of Pa claim 1 solved. The sub-claims describe before partial configurations of the process. The invented Antibodies produced according to the method are before in a pharmaceutical preparation as therapeutic agents used.
Das Verfahren bietet zum ersten Mal die Möglichkeit, mensch liche Antikörper in der Zellkultur in solchen Mengen herzu stellen, daß eine Reinigung der Antikörper und deren medizini sche Anwendung möglich ist.For the first time, the process offers the possibility of human antibodies in cell culture in such quantities make a cleaning of the antibodies and their medicinal cal application is possible.
Nachfolgend wird die Erfindung anhand bevorzugter Ausgestal tungen näher erläutert; die Erfindung-ist jedoch nicht hierauf beschränkt. Weitere Ausführungsformen sind vom Fachmann ohne erfinderisches Zutun durchführbar. The invention is illustrated below with the aid of a preferred embodiment explained in more detail; however, the invention is not based on this limited. Other embodiments are without a person skilled in the art inventive intervention feasible.
Erfindungsgemäß werden somit zunächst Antikörper produzierende B-Zellen mit einem Epstein-Barr-Virus oder einem entweder na türlich vorkommenden oder gentechnisch hergestellten Derivat des Epstein-Barr-Virus infiziert. In einer weiteren Ausfüh rungsform der Erfindung wird ein Vektor verwendet, der zumin dest diejenigen Sequenzen des Epstein-Barr-Virus aufweist, die zur Immortalisierung menschlicher B-Zellen notwendig sind. Bevorzugt werden primäre menschliche B-Zellen infiziert.According to the invention, antibodies are thus first produced B cells with an Epstein-Barr virus or either na naturally occurring or genetically engineered derivative of the Epstein-Barr virus. In another version Form of the invention, a vector is used, the at least those sequences of the Epstein-Barr virus that are necessary to immortalize human B cells. Primary human B cells are preferably infected.
Epstein-Barr-Virus (EBV) ist ein lymphotropes Herpes-Virus, das für die infektiöse Mononukleose verantwortlich ist. Die infektiöse Mononukleose, auch Pfeiffer-Drüsenfieber genannt, ist eine lymphoproliferative Erkrankung, die zu einer Hyper plasie und Hypertrophie des lymphatischen Gewebes mit charak teristischen Blutbildveränderungen führt. EBV infiziert primä re, ruhende B-Zellen, wodurch in vitro unbegrenzt proliferie rende lymphoblastoide Zellinien (LCLs) entstehen. Dieser Pro zeß wird auch als Immortalisierung oder Transformation be zeichnet. EBV wird weiterhin mit verschiedenen menschlichen Erkrankungen assoziiert, zu denen das Burkitt-Lymphom (BL), Nasopharynx-Karzinome, Lymphome immunkompromittierter Personen und Morbus Hodgkin (vgl. Übersichten in Miller, 1990; Klein, 1994; Masucci und Ernberg, 1994) gehören.Epstein-Barr virus (EBV) is a lymphotropic herpes virus, that is responsible for infectious mononucleosis. The infectious mononucleosis, also called Pfeiffer glandular fever, is a lymphoproliferative disorder that leads to hyper plasticity and hypertrophy of the lymphatic tissue with charak changes in blood count. EBV primarily infects right, resting B cells, which proliferates indefinitely in vitro lymphoblastoid cell lines (LCLs) are formed. That pro zeß is also used as immortalization or transformation draws. EBV will continue to work with various human Diseases associated with Burkitt's lymphoma (BL), Nasopharynx carcinomas, lymphomas of immunocompromised people and Hodgkin's disease (see reviews in Miller, 1990; Klein, 1994; Masucci and Ernberg, 1994) belong.
In LCL-Zellen wird nur ein Teil der viralen Gene exprimiert. Die exprimierten Virusgene kodieren für sechs Kernproteine, drei Membranproteine und zwei kleine Kern-RNAs, die nicht po lyadenyliert sind. Die minimale Anzahl von Genen, die für die Transformation erforderlich ist, ist bis heute unbekannt. Das EBNA2-Gen ist in dem transformationsdefekten Virus, der von der Burkitt-Lymphomzellinie P3HR1 produziert wird, deletiert. Es kommt in natürlichen Isolaten in zwei allelen Formen vor (EBNA2A und EBNA2B), die in etwa 50% Homologie zeigen (Zimber et al., 1986). EBNA2 ist zusammen mit EBNA-LP das erste Virus gen, das nach Infektion primärer B-Zellen exprimiert wird, und es ist ein Transkriptionsaktivator sowohl von latenten Virus als auch von latenten Zellgenen (CD21, CD23 und c-fgr) (Calender et al., 1987; Wang et al., 1987; Cordier et al., 1990; Ghosh und Kieff, 1990; Fahraeus et al., 1990; Abbot et al., 1990; Knutson, 1990; Woisetschlaeger et al., 1991; Zimber-Strobl et al., 1991, 1993; Sung et al., 1991; Jin und Speck, 1992; Ling et al., 1993; Laux et al., 1994a; Meitinger et al., 1995).Only part of the viral genes are expressed in LCL cells. The expressed virus genes code for six core proteins, three membrane proteins and two small nuclear RNAs that are not po are lyadenylated. The minimum number of genes for the Transformation is required is still unknown. The EBNA2 gene is in the transformation defective virus by of the Burkitt lymphoma cell line P3HR1 is deleted. It occurs in natural isolates in two allelic forms (EBNA2A and EBNA2B), which show approximately 50% homology (Zimber et al., 1986). Together with EBNA-LP, EBNA2 is the first virus gene that is expressed after infection of primary B cells, and it is a transcriptional activator of both latent virus as well as latent cell genes (CD21, CD23 and c-fgr) (Calender et al., 1987; Wang et al., 1987; Cordier et al., 1990; Ghosh and Kieff, 1990; Fahraeus et al., 1990; Abbot et al., 1990; Knutson, 1990; Woisetschlaeger et al., 1991; Zimber-Strobl et al., 1991, 1993; Sung et al., 1991; Jin and Bacon, 1992; Ling et al., 1993; Laux et al., 1994a; Meitinger et al., 1995).
Es konnte gezeigt werden, daß EBNA2 seine Transaktivierungs funktion zumindest teilweise durch Bindung an ein ubiquitär exprimiertes Zellgen, RBP-J, das an die DNA in sequenzspezi fischer Weise bindet (Zimber-Strobl et al., 1994; Henkel et al., 1994; Grossmann et al., 1994; Waltzer et al, 1994) und durch Wechselwirkung mit Transkriptionsfaktorender ets-Genfa milie (SPi-1, PU-1) (Laux et al., 1994b; und Johannsen et al., 1995) bewirkt. Obwohl die Transformation primärer B-Zellen in vitro strikt von EBNA2 abhängt (Cohen et al., 1989; Hammer schmidt und Sugden, 1989; Kempkes et al., 1995), scheint EBNA2 in BL-Tumoren (Rowe et al., 1986), beim Morbus Hodgkin (Kana varos et al., 1993) und in Nasopharynx-Karzinomen (Fahraeus et al., 1988; Young et al., 1988) nicht exprimiert zu werden, weshalb seine Rolle bei der Entstehung dieser Erkrankungen in vivo unklar ist.It could be shown that EBNA2 is its transactivation function at least in part by binding to an ubiquitous expressed cell gene, RBP-J, which binds to the DNA in sequence-spec fischer manner (Zimber-Strobl et al., 1994; Henkel et al., 1994; Grossmann et al., 1994; Waltzer et al, 1994) and through interaction with transcription factors of the ets gene milie (SPi-1, PU-1) (Laux et al., 1994b; and Johannsen et al., 1995). Although the transformation of primary B cells into vitro strictly depends on EBNA2 (Cohen et al., 1989; Hammer Schmidt and Sugden, 1989; Kempkes et al., 1995) appears to be EBNA2 in BL tumors (Rowe et al., 1986), in Hodgkin's disease (Kana varos et al., 1993) and in nasopharyngeal carcinomas (Fahraeus et al., 1988; Young et al., 1988) not to be expressed, which is why its role in causing these diseases in vivo is unclear.
Um die Rolle von EBNA2 bei der Transformation von B-Zellen besser zu verstehen, wurde ein konditionales System entwickelt, bei dem die Funktion des EBNA2-Proteins reversibel an- und ausschaltbar ist. Unter Verwendung des von Picard et al. (1988) und Eilers et al. (1989) entwickelten induzierbaren Systems wurde die Hormonbindungsdomäne des Östrogenrezeptors mit dem N- oder C-Terminus von EBNA2 fusioniert, wodurch die Funktion des EBNA2-Proteins nunmehr davon abhängt, ob Östrogen vorhanden ist oder nicht. Wie erwartet, bewirken die chimeren EBNA2-Proteine ihre transaktivierende Funktion auf virale und zelluläre Gene nur in Gegenwart von Östrogen.To the role of EBNA2 in the transformation of B cells Understanding better became a conditional system developed in which the function of the EBNA2 protein is reversible can be switched on and off. Using the Picard et al. (1988) and Eilers et al. (1989) developed inducible Systems became the hormone binding domain of the estrogen receptor fused to the N or C terminus of EBNA2, making the Function of the EBNA2 protein now depends on whether estrogen is present or not. As expected, the chimeras do EBNA2 proteins have their transactivating function on viral and cellular genes only in the presence of estrogen.
Erfindungsgemäß wurde überraschend gefunden, daß durch das EBNA2-Protein die Expression von Oberflächen-IgM und die Transkription des Ig-µ-Locus sehr effizient verringert wird. EBNA2 wirkt somit als negativer Regulator der Ig-µ-Transkrip tion.According to the invention, it was surprisingly found that the EBNA2 protein expression of surface IgM and the Transcription of the Ig-µ locus is reduced very efficiently. EBNA2 thus acts as a negative regulator of the Ig-µ transcript tion.
Die Erfindung wird im folgenden anhand von Ausführungsbeispie len mit Hilfe der Figuren näher erläutert. Dabei zeigen die Fig. 1 und 2 exemplarisch die Erhöhung der immunglobulin protein-Produktion durch Abschaltung von EBNA2. Die Fig. 3 und 4 zeigen entsprechend die Immunglobulin-RNA-Produktion.The invention is explained below with reference to Ausführungsbeispie len with the help of the figures. Here, FIGS. 1 and 2 show examples of increasing the immunoglobulin protein production by switching off EBNA2. FIGS. 3 and 4 respectively show the immunoglobulin RNA production.
Die Erfindung beruht auf der überraschenden Tatsache, daß Ep stein-Barr-Virus-infizierte B-Zellen nur geringe Mengen Immunglobulin produzieren, die Produktion nach Inaktivierung des EBNA2-Gens aber um das mindestens 100-fache steigt.The invention is based on the surprising fact that Ep B-cells infected with stone-Barr virus only small amounts Produce immunoglobulin, production after inactivation of the EBNA2 gene, however, increases by at least 100 times.
Die Erfindung wird in den Ausführungsbeispielen anhand einer IgM-produzierenden B-Zellinie näher beschrieben. Die Erfindung ist jedoch nicht auf eine derartige, IgM-produzierende B-Zel linie beschränkt. Erfindungsgemäß können auch B-Zellen durch EBV immortalisiert werden, die andere Immunglobulinklassen exprimieren, beispielsweise IgG und IgA.The invention is in the exemplary embodiments with reference to a IgM-producing B cell line described in more detail. The invention is not, however, of such an IgM-producing B cell line limited. According to the invention, B cells can also pass through Immobilized EBV, the other immunoglobulin classes express, for example IgG and IgA.
Ursprung der verwendeten B-Zellen sind im Normalfall Menschen mit einem hohen Antikörpertiter für das gewünschte Antigen. Zellen werden vorzugsweise aus dem peripheren Blut gewonnen, und B-Zellen werden nach Standardverfahren angereichert und mit einem Epstein-Barr-Virus-abgeleiteten Virus, welches ein konditionales EBNA2 exprimiert, infiziert. Während der Infek tion und Vermehrung der B-Zellen muß die EBNA2-Funktion akti viert sein. Das von Kempkes et al. (1995) EMBO J. 14, 88-96, beschriebene Virus ist zur Infektion besonders geeignet (vgl. Fig. 5) und die nachfolgende Beschreibung. Zur Aktivierung der EBNA2-Funktion muß Östrogen zum Kulturmedium gegeben werden, um eine konstitutive EBNA2-Funktion zu gewährleisten. Die im mortalisierten B-Zellen werden expandiert und mehrmals mit Östrogenhaltigem Medium verdünnt, bis die gewünschte Zellzahl erreicht ist. Die Zellen werden in Mikrotiterplatten in gerin ger Zellzahl ausplattiert und erneut expandiert. Der Überstand der Zellen wird auf die gewünschten spezifischen Antikörper hin untersucht. B-Zellen, die Antikörper mit hoher Spezifität produzieren, werden als Grundlage der Antikörperherstellung verwendet.The origin of the B cells used are usually people with a high antibody titer for the desired antigen. Cells are preferably obtained from peripheral blood, and B cells are enriched by standard methods and infected with an Epstein-Barr virus-derived virus that expresses conditional EBNA2. The EBNA2 function must be activated during infection and multiplication of the B cells. The Kempkes et al. (1995) EMBO J. 14, 88-96, the virus described is particularly suitable for infection (cf. FIG. 5) and the description below. To activate the EBNA2 function, estrogen must be added to the culture medium to ensure a constitutive EBNA2 function. The mortalized B cells are expanded and diluted several times with medium containing estrogen until the desired number of cells is reached. The cells are plated out in microtiter plates in a small number of cells and expanded again. The supernatant of the cells is examined for the desired specific antibodies. B cells that produce antibodies with high specificity are used as the basis for antibody production.
In diesen B-Zellklonen wird die EBNA2-Aktivität durch Auswa schen des Östrogens abgeschaltet. Andere Methoden der Entfer nung des Östrogens sind ebenfalls anwendbar. Alternativ kann die Aktivität des EBNA2-Gens auch mit der Hormonbindungsdomäne des Androgenrezeptors oder einer anderen Hormonbindungsdomäne oder auf transkriptioneller Ebene durch Verwendung konditiona ler Promotoren für das EBNA2-Gen reguliert werden. Mit Ab schaltung der EBNA2-Funktion wird die Immunglobulin-Produktion in den B-Zellen kontinuierlich gesteigert und ist nach 2 Tagen ca. 100-fach angestiegen. Nach 4 Tagen beginnt ein großer Teil der Zellen zu sterben.In these B cell clones, the EBNA2 activity is determined by Auswa switched off estrogen. Other methods of removal estrogen are also applicable. Alternatively, you can the activity of the EBNA2 gene also with the hormone binding domain of the androgen receptor or another hormone binding domain or at the transcriptional level by using conditiona promoters for the EBNA2 gene are regulated. With Ab Switching the EBNA2 function is the immunoglobulin production in the B cells increased continuously and is after 2 days increased about 100 times. A large part begins after 4 days of the cells to die.
Nachfolgend wird die Konstruktion eines Plasmids beschrieben, das zur konditionalen Expression des EBNA2-Gens oder einem Derivat hiervon verwendbar ist. Die Erfindung ist jedoch nicht auf das hier beschriebene Plasmid beschränkt. Erfindungsgemäß können auch andere Vektoren verwendet werden, mit denen das EBNA2-Gen konditional exprimierbar ist, d. h. bei der Immorta lisierung der B-Zellen zumindest teilweise angeschaltet ist und vor Gewinnung der Antikörper zumindest teilweise abge schaltet ist. Bevorzugt ist das EBNA2-Gen zur Immortalisierung im wesentlichen angeschaltet und zur Gewinnung der Antikörper im wesentlichen abgeschaltet.The construction of a plasmid is described below, that for the conditional expression of the EBNA2 gene or a Derivative thereof can be used. However, the invention is not limited to the plasmid described here. According to the invention other vectors can be used with which the EBNA2 gene is conditionally expressible, i. H. at the Immorta lization of the B cells is at least partially switched on and at least partially abge before obtaining the antibodies is switched. The EBNA2 gene is preferred for immortalization essentially turned on and to raise the antibodies essentially turned off.
Die Hormonbindungsdomäne des Östrogenrezeptors wurde entweder an den N- oder C-Terminus des offenen Leserahmens (open reading frame = ORF) von EBNA2 angehängt, wodurch die Plasmide ER/EBNA2 und EBNA2/ER entstanden. Beide Plasmide wiesen die Gesamtstruktur des Mini-EBV p554 auf und exprimierten Wildtyp- EBNA2, EBNA-LP und den offenen Leserahmen vom BHRF1 (Hammerschmidt und Sugden, 1989). Das N-terminale Fusionskon strukt wurde durch Einführung einer EcoRI-Schnittstelle unmit telbar 5′ an den ORF von EBNA2 durch in vitro-Mutagenese ge bildet. Das für die Hormonbindungsdomäne kodierende Genfrag ment wurde durch die Polymerasekettenreaktion (PCR) unter Ver wendung von HE14 (Kumar et al., 1986) als Matrize, flankiert von den EcoRI-Erkennungsstellen und ligiert in die EcoRI-Erken nungsstelle vor den ORF von EBNA2, gebildet. Dieses PCR-Frag ment umfaßte die von HE14 bereitgestellte Kozak-Sequenz, je doch nicht das Stop-Codon des Östrogenrezeptors. Das C-termi nale Fusionskonstrukt wurde durch Insertion von BglII- und EcoRI-Erkennungsstellen unmittelbar von dem Stop-Codon von EBNA2 durch in vitro-Mutagenese erzeugt. In die BglII/EcoRI- Erkennungsstellen am EBNA2-Ende wurde ein BamHI-EcoRI-Fragment von HE14, das für die Hormonbindungsstelle des Östrogenrezep tors kodiert, ligiert. Die nachfolgenden Klonierungsschritte ergaben den EBV-Stamm B95.8 mit den Koordinaten 13944-54364 (vgl. Baer et al., 1984) mit zwei Abänderungen: die NotI-Repe ats waren deletiert und nur zwei BamHI W-Repeats waren inser tiert.The hormone binding domain of the estrogen receptor was either at the N or C terminus of the open reading frame (open reading frame = ORF) from EBNA2, causing the plasmids ER / EBNA2 and EBNA2 / ER emerged. Both plasmids showed the Overall structure of the mini EBV p554 on and expressed wild-type EBNA2, EBNA-LP and the open reading frame from BHRF1 (Hammerschmidt and Sugden, 1989). The N-terminal fusion con structure was introduced immediately by introducing an EcoRI interface telbar 5 ′ to the ORF of EBNA2 by in vitro mutagenesis forms. The gene question coding for the hormone binding domain ment was by the polymerase chain reaction (PCR) with Ver using HE14 (Kumar et al., 1986) as a template from the EcoRI recognition sites and ligated into the EcoRI orks in front of the ORF of EBNA2. This PCR question ment included the Kozak sequence provided by HE14, each but not the stop codon of the estrogen receptor. The C-termi nale fusion construct was by insertion of BglII and EcoRI recognition sites immediately from the stop codon of EBNA2 generated by in vitro mutagenesis. In the BglII / EcoRI- A BamHI-EcoRI fragment was recognized at the EBNA2 end of HE14, which is for the hormone binding site of the estrogen recipe tors coded, ligated. The subsequent cloning steps gave the EBV strain B95.8 with the coordinates 13944-54364 (see Baer et al., 1984) with two changes: the NotI-Repe ats were deleted and only two BamHI W repeats were inserted animals.
Die Fig. 5 zeigt die Strategie zur Expression konditionaler EBNA2-Mutanten in wachstumstransformierten B-Zellen. FIG. 5 shows the strategy for the expression of conditional EBNA2 mutants in growth-transformed B cells.
(A) der Fig. 5 zeigt Mini-EBV-Plasmide, die aus in cis wirken den Elementen bestehen, die zur Vermehrung dieser Plasmide in allen Phasen des Lebenszyklus des Epstein-Barr-Virus essen tiell sind. Diese Mini-EBV-Plasmide tragen den Replikations ursprung des Plasmids (oriP), der für die Beibehaltung der EBV-Plasmide in den Targetzellen notwendig ist, den lytischen Replikationsursprung (oriLyt), der bei Induktion der lytischen Replikation des Virus eine Amplifikation mit hoher Kopienan zahl ermöglicht, und die terminalen Repeats (TR), die eine Verpackung der Mini-EBV-Plasmide in Virionen ermöglichen. Die Hormonbindungsdomäne des Östrogenrezeptors (weiße Kästchen) wurden entweder an den C- oder N-Terminus des offenen Leserahmens des Wildtyp-EBNA2 (schwarze Kästchen) angehängt, wodurch die EBNA2/ER- bzw. ER/EBNA2-Mutanten entstanden. Die EBNA2-Expressionskassetten werden von EBV-Sequenzen flankiert, den BamHI-Restriktionsfragmenten C, W, Y und H des EBV-Genoms, die teilweise die Genomorganisation der EBV-DNA darstellen und die die im nicht-transformierenden P3HR1-EBV-Stamm vorhandene Deletion umfassen (ΔP3HR1, offener Balken in B). EBNA2 und EBNA2-Mutanten können von zwei alternativen Promotoren trans kribiert werden, die in den C- oder W-Fragmenten lokalisiert sind.(A) of FIG. 5 shows mini-EBV plasmids which consist of elements which act in cis and which are essential for the propagation of these plasmids in all phases of the life cycle of the Epstein-Barr virus. These mini-EBV plasmids carry the replication origin of the plasmid (oriP), which is necessary for the retention of the EBV plasmids in the target cells, the lytic origin of replication (oriLyt), which, upon induction of the lytic replication of the virus, amplifies with high copies number, and the terminal repeats (TR), which enable the packaging of the mini-EBV plasmids in virions. The hormone binding domain of the estrogen receptor (white boxes) were attached to either the C or N terminus of the open reading frame of the wild-type EBNA2 (black boxes), resulting in the EBNA2 / ER and ER / EBNA2 mutants, respectively. The EBNA2 expression cassettes are flanked by EBV sequences, the BamHI restriction fragments C, W, Y and H of the EBV genome, which partly represent the genomic organization of the EBV DNA and the deletion present in the non-transforming P3HR1-EBV strain include (ΔP3HR1, open bar in B). EBNA2 and EBNA2 mutants can be transcribed from two alternative promoters located in the C or W fragments.
Die Fig. 5 (B) zeigt die EBV-Karte mit den in wachstumstrans formierten B-Zellen transkribierten EBNAs und LMPs und die Lokalisierung ihrer Promotoren in bezug auf die BamHI-Restrik tionskarte des EBV-Stammes B95.8.The Fig. 5 (B) shows the EBV-card with a brief break in the growth trans B cells transcribed EBNAs and LMPs and the localization of their promoters with respect to the BamHI-restrictive tion map of the EBV strain B95.8.
Bei den östrogenabhängigen Zellinien ER/EB2-3 und ER/EB2-5 handelt es sich um lymphoblastoide Zellinien (LCLs), die durch Coinfektion primärer B-Zellen mit P3HR1-Virus und einem Mini- EBV-Plasmid, das ER/EBNA2 exprimiert und das den EBNA2-Defekt des P3HR1-Virus in trans komplementiert, etabliert wurden (Kempkes et al., 1995). Die östrogenunabhängige EB2-2-Zellinie wurde parallel hierzu unter Verwendung von Wildtyp-EBNA2 er zeugt, um den EBNA2-Defekt des P3HR1-Virus zu komplementieren. BL41 ist eine EBV-negative BL-Zellinie mit einer t(8;14)-Translokation (Lenoir et al., 1985), BL41/P3HR1 und BL41/B95-8 sind BL41-Zellen, die mit P3HR1- bzw. B95-8-Virus stabil infi ziert (umgewandelt) wurden (Calender et al, 1987). BJAB ist eine EBV-negative B-Lymphomzellinie (Klein et al, 1974); BJAB/P3 ist eine BJAB-Zellinie, die mit P3HR1-Virus stabil infiziert ist. P3HR1 ist ein Einzelzellklon der EBV-positiven B-Zellinie Jÿoye mit einer t(8;14)-Translokation (Hinuma et al, 1967). Jÿoye produziert ein EBNA2-kompetentes und die P3HR1-Zellen ein EBNA2-defektes Virus (Bornkamm et al., 1982). Die ER/EBNA2 exprimierenden Lymphomzellinien sind bei Kempkes et al. beschrieben. Alle Zellinien wurden in RPMI1640-Medium kultiviert, das mit 10% fötalem Kälberserum, 100 U/ml Penicil lin und Streptomycin und 1 mM Pyruvat supplementiert war. β-Östradiol (Merck, Darmstadt) wurde zum Zellkulturmedium in einer Endkonzentration von 1 µM zugegeben.For the estrogen-dependent cell lines ER / EB2-3 and ER / EB2-5 are lymphoblastoid cell lines (LCLs) that pass through Coinfection of primary B cells with P3HR1 virus and a mini EBV plasmid that expresses ER / EBNA2 and that the EBNA2 defect of the P3HR1 virus in trans complemented, were established (Kempkes et al., 1995). The estrogen-independent EB2-2 cell line was performed in parallel using wild-type EBNA2 testifies to complement the EBNA2 defect of the P3HR1 virus. BL41 is an EBV-negative BL cell line with at (8; 14) translocation (Lenoir et al., 1985), BL41 / P3HR1 and BL41 / B95-8 are BL41 cells that are stable with P3HR1 or B95-8 virus were decorated (converted) (Calender et al, 1987). BJAB is an EBV negative B lymphoma cell line (Klein et al, 1974); BJAB / P3 is a BJAB cell line that is stable with P3HR1 virus is infected. P3HR1 is a single cell clone of EBV-positive B-cell line Jÿoye with at (8; 14) translocation (Hinuma et al, 1967). Jÿoye produces an EBNA2-competent and the P3HR1 cells an EBNA2-defective virus (Bornkamm et al., 1982). The ER / EBNA2 expressing lymphoma cell lines are from Kempkes et al. described. All cell lines were in RPMI1640 medium cultured with 10% fetal calf serum, 100 U / ml Penicil lin and streptomycin and 1 mM pyruvate was supplemented. β-estradiol (Merck, Darmstadt) became the cell culture medium in a final concentration of 1 µM was added.
Zur Durchführung der FACS-Analyse wurden 1 × 10⁶ Zellen mit einem Überschuß an unmarkierten monoklonalen Antikörpern der Maus, die CD21 (BL13) und Oberflächen-IgM (AF6) (Dianova, Ham burg) erkennen, inkubiert. Mit FITC (Fluorescein) konjugierte Ziege-anti-Maus F(ab)₂-Fragmente (DAKO, Hamburg) wurden zur Färbung positiver Zellen als sekundärer Antikörper verwendet. Tote Zellen wurden identifiziert und nach Propidiumiodidfär bung (0,1 µg/ml) von der Analyse ausgeschlossen. Die Proben wurden unter Verwendung eines FACSSCAN-Geräts (Becton und Dickinson) analysiert. Der 3-H-Thymidineinbau-Test wurde wie bei Kempkes et al., 1995 beschrieben durchgeführt.To carry out the FACS analysis, 1 × 10⁶ cells were used an excess of unlabelled monoclonal antibodies to the Mouse, the CD21 (BL13) and surface IgM (AF6) (Dianova, Ham castle) recognize, incubated. Conjugated with FITC (fluorescein) Goat anti-mouse F (ab) ₂ fragments (DAKO, Hamburg) were used Staining positive cells used as a secondary antibody. Dead cells were identified and stained for propidium iodide (0.1 µg / ml) excluded from the analysis. Samples were made using a FACSSCAN device (Becton and Dickinson) analyzed. The 3-H-thymidine incorporation test was like in Kempkes et al., 1995.
Die Northern-Blot-Analysen wurden wie bei Eick und Bornkamm, 1989 beschrieben durchgeführt. Als Igp-Gen-Sonden wurde das 1,2 kb große EcoRI/EcoRI-Fragment und das 0,9 kb EcoRI/SacI- Fragment der konstanten Region, beschrieben in Eick et al. (1985), verwendet.The Northern blot analyzes were performed as for Eick and Bornkamm, Described in 1989. This was called the Igp gene probe 1.2 kb EcoRI / EcoRI fragment and the 0.9 kb EcoRI / SacI Constant region fragment described in Eick et al. (1985).
In den oben beschriebenen, konditional transformierten lympho blastoiden Zellen wurde die Expression von Oberflächenmarkern vor und nach Entfernung von Östrogen untersucht.In the conditionally transformed lympho described above blastoid cells became the expression of surface markers examined before and after removal of estrogen.
Die Fig. 1 zeigt eine Flußzytometrieanalyse von mit Östrogen behandelten (weiße Histogramme) und an Östrogen verarmten (schwarze Histrogramme) ER/EB2-5-Zellen nach CD21-, CD23- und Oberflächen-IgM-Färbung. Figure 1 shows a flow cytometric analysis of estrogen-treated (white histograms) and estrogen-depleted (black histograms) ER / EB2-5 cells after CD21, CD23 and surface IgM staining.
Die Fig. 1 zeigt insbesondere die Menge an IgM-Antikörpern, die in Anwesenheit (weiße Fläche) und Abwesenheit (schwarze Fläche) von EBNA2-Aktivität von einer EBV-immortalisierten B- Zelle nach 2 Tagen produziert wird. Vergleichbare Ergebnisse wurden nach Immortalisierung primärer B-Zellen mit dem oben genannten Virus für zwei weitere Zellinien erhalten. Figure 1 shows in particular the amount of IgM antibodies which are produced in the presence (white area) and absence (black area) of EBNA2 activity by an EBV-immortalized B cell after 2 days. Comparable results were obtained after immortalizing primary B cells with the above-mentioned virus for two further cell lines.
Die Fig. 1 zeigt, daß die CD23- und in einem geringeren Ausmaß die CD21-Expression durch Abschaltung der Funktion von EBNA2 durch Entfernung des Östrogens abnahm. Gleichzeitig nahm je doch die IgM-Expression stark zu. Die Zunahme der IgM-Expres sion und die Abnahme der CD21- und CD23-Expression waren re versibel, da sowohl die CD21- als auch die CD-23-Expression zunahmen und die IgM-Expression abnahm, wenn anschließend Östrogen zugegeben wurde. Die Ergebnisse sind hier nicht im einzelnen wiedergegeben. Figure 1 shows that CD23 and, to a lesser extent, CD21 expression decreased by shutting down the function of EBNA2 by removing the estrogen. At the same time, however, IgM expression increased sharply. The increase in IgM expression and the decrease in CD21 and CD23 expression were reversible since both CD21 and CD-23 expression increased and IgM expression decreased when estrogen was subsequently added. The results are not shown in detail here.
Die Abnahme des Oberflächen-IgMs könnte entweder durch EBNA2 selbst oder durch andere virale, stromabwärts gelegene Targets (z. B. die viralen latenten Membranproteine), die durch EBNA2 induziert werden, vermittelt werden. Um zwischen diesen zwei Möglichkeiten zu unterscheiden, wurde das das ER/EBNA2-Fu sionsprotein kodierende Gen stabil in die EBV-negativen B-Zell-Lymphomlinien BL41 und BJAB und zum Vergleich auch in P3HR1-Zellen eingeführt. In den stabil transfizierten Zellini en war die Aktivierung des TP1- oder des LMP1-Promotors strikt an das Vorliegen von Östrogen gebunden, was zeigt, daß das ER/ENBA2-Protein tatsächlich in diesen Zellen funktionell ist. Eine FACS-Analyse vor und nach Zugabe von Östrogen zeigte eine starke Abnahme der sIgM-Expression bei Zugabe von Östrogen (Fig. 2).The decrease in surface IgM could be mediated either by EBNA2 itself or by other viral downstream targets (e.g. the viral latent membrane proteins) induced by EBNA2. In order to differentiate between these two possibilities, the gene coding for the ER / EBNA2 fusion protein was stably introduced into the EBV-negative B cell lymphoma lines BL41 and BJAB and for comparison also into P3HR1 cells. In the stably transfected cell lines, the activation of the TP1 or LMP1 promoter was strictly linked to the presence of estrogen, which shows that the ER / ENBA2 protein is indeed functional in these cells. A FACS analysis before and after adding estrogen showed a sharp decrease in sIgM expression when adding estrogen ( FIG. 2).
Hieraus kann geschlossen werden, daß die Abnahme der sIgM-Ex pression eine Funktion von EBNA2 ist und keine Funktion eines anderen viralen Targetgens.From this it can be concluded that the acceptance of the sIgM-Ex pression is a function of EBNA2 and not a function of other viral target gene.
Es wurden Northern-Blot-Analysen durchgeführt, um herauszufin den, bei welchem Niveau die IgM-Expression durch EBNA2 nach unten reguliert wird. Durch die Aufnahme von RNA aus Paarzellinien, die sich durch das Vorliegen oder das Fehlen des Wildtyp EBNA2-Gens unterscheiden, ergab die Northern-Blot- Analyse weiterhin eine Antwort auf die Frage, ob die IgM-Ab nahme nicht nur durch das EBNA2-Östrogen-Rezeptorfusionspro tein bewirkt wird, sondern auch durch das Wildtyp-EBNA2. Die Paarzellinien umfaßten P3HR1, das ein EBNA2-defekter Virus produziert, und seine Elternzellinie Jÿoye mit einem funktio nellen EBNA2-Gen (EBNA2B-Allel) und BL41-Zellen, die mit P3HR1-Virus stabil infiziert/umgewandelt (konvertiert) waren (BL41/p3HR1) oder dem transformationskompetenten, EBNA2-Wild typ-positiven B95-8-Virus mit dem EBNA2-Allel. Wie die Fig. 3 zeigt, wurde die Oberflächen-Expression durch EBNA2 durch Ab nahme der Igµ-RNA nach unten reguliert.Northern blot analyzes were performed to determine the level at which IgM expression is downregulated by EBNA2. By incorporating RNA from pair cell lines that differ in the presence or absence of the wild-type EBNA2 gene, the Northern blot analysis further provided an answer to the question whether the IgM decrease was not only due to the EBNA2 estrogen Receptor fusion protein is effected, but also by the wild-type EBNA2. The pair cell lines included P3HR1, which produces an EBNA2-defective virus, and its parent cell line Jÿoye with a functional EBNA2 gene (EBNA2B allele) and BL41 cells, which were stably infected / converted (converted) with P3HR1 virus (BL41 / p3HR1) or the transformation-competent, EBNA2 wild type positive B95-8 virus with the EBNA2 allele. As shown in FIG. 3, the surface expression by EBNA2 was regulated downwards by taking the Igμ-RNA.
Die Fig. 2 zeigt die Menge an IgM-Antikörpern, die in Anwe senheit (weiße Fläche) und Abwesenheit (schwarze Fläche) von ENBA2-Aktivität von zwei Burkitt-Lymhom-Zellinien nach 2 Tagen produziert wird. Figure 2 shows the amount of IgM antibodies produced in the presence (white area) and absence (black area) of ENBA2 activity by two Burkitt-Lymhom cell lines after 2 days.
Die Fig. 2 zeigt insbesondere die Suppression von Zelloberflä chen-IgM durch EBNA2 in B-Lymphomzellinien. ER/EBNA2-transfi zierte Zellen (BJAB/K3, BL41/K3 und P3HR1/3B6) wurden 2 Tage lang in Gegenwart von (weiße Histogramme) oder ohne (schwarze Histogramme) 1 µM β-Östradiol kultiviert und dann auf Zelloberflächen-CD21 und Oberflächen-IgM-Expression durch FACS-Analyse untersucht. 1 × 10⁶ Zellen wurden mit einem Über schuß an unmarkierten monoklonalen Antikörpern der Maus inku biert, die Human-CD21 (BL13) oder IgM (AF6) erkennen. FITC- konjugierte Ziege-anti-Maus F(ab)₂-Fragmente wurden als sekun därer Antikörper zur Färbung von positiven Zellen verwendet. Kontrollzellen wurden nur mit sekundären Antikörpern markiert; sie zeigten jedoch keinerlei Veränderungen auf Östrogenzugabe (Daten nicht gezeigt). Tote Zellen wurden identifiziert und aus der Analyse nach Propidiumiodidfärbung (0,1 µg/ml) ausge schlossen. FIG. 2 shows in particular the suppression of cell surface IgM by EBNA2 in B lymphoma cell lines. ER / EBNA2-transfected cells (BJAB / K3, BL41 / K3 and P3HR1 / 3B6) were cultured for 2 days in the presence of (white histograms) or without (black histograms) 1 µM β-estradiol and then on cell surface CD21 and Surface IgM expression examined by FACS analysis. 1 × 10⁶ cells were incubated with an excess of unlabelled mouse monoclonal antibodies that recognize human CD21 (BL13) or IgM (AF6). FITC-conjugated goat anti-mouse F (ab) ₂ fragments were used as secondary antibodies for staining positive cells. Control cells were only labeled with secondary antibodies; however, they showed no changes in estrogen addition (data not shown). Dead cells were identified and excluded from the analysis after propidium iodide staining (0.1 µg / ml).
Die Fig. 3 und 4 zeigen in analoger Weise die Menge der entsprechenden Immunglobulin-RNAs. FIGS. 3 and 4 show in an analogous manner, the amount of the corresponding immunoglobulin RNAs.
Die Fig. 3 zeigt speziell die Suppression der Expression der schweren Kette von Ig-u durch EBNA2 in B-Lymphomzellen und LCLs. Gesamtzell-RNA wurde aus JiJoye, P3HR1, BL41, BL41/P3HR1, BL41/B95-8 und ER/ENBA2-transfizierten Zellen, die zwei Tage lang mit oder ohne Östrogene (A) kultiviert worden waren, oder aus LCL-Zellinien, die das ER/EBNA2-Protein (ER/EB2-3 und ER/EB2-5) oder das Wildtyp EBNA2-Protein (EB2-2) exprimieren, wobei die Zellinien mit Östrogen behandelt worden waren oder vier Tage lang an Östrogen verarmt wurden (B), iso liert, und die Zellen wurden in bezug auf die schwere Kette von Ig-µ-, die leichte Kette von Ig- und GAPDH-RNA durch Northern-Blot-Analyse analysiert. Figure 3 specifically shows the suppression of Ig-u heavy chain expression by EBNA2 in B-lymphoma cells and LCLs. Whole cell RNA was derived from JiJoye, P3HR1, BL41, BL41 / P3HR1, BL41 / B95-8 and ER / ENBA2-transfected cells that had been cultured for two days with or without estrogens (A) or from LCL cell lines that express the ER / EBNA2 protein (ER / EB2-3 and ER / EB2-5) or the wild-type EBNA2 protein (EB2-2) after the cell lines had been treated with estrogen or depleted in estrogen for four days (B ), isolated and the cells were analyzed for the heavy chain of Ig-µ-, the light chain of Ig- and GAPDH-RNA by Northern blot analysis.
Die Fig. 4 zeigt die Coregulation der Ig-µ- und der c-myc-Gen expression in ER/EBNA2-transfizierten BL-Zellen. P3HR1/3B6 (A)-Zellen wurden mit Östrogen (+) für die angegebene Zeitdau er behandelt und auf Expression von Ig-µ- und c-myc-RNA durch Northern-Analyse untersucht. Am unteren Rand des Bildes sind die mit Ethidiumbromid gefärbten Gele gezeigt. In östrogenab hängigen LCLs werden die IG-µ- und die c-myc-Expression durch Östrogen einander entgegengesetzt reguliert (B). Fig. 4 shows the co-regulation of the Ig-µ and the c-myc gene expression in ER / EBNA2-transfected BL cells. P3HR1 / 3B6 (A) cells were treated with estrogen (+) for the stated time period and examined for expression of Ig-µ and c-myc RNA by Northern analysis. The gels stained with ethidium bromide are shown at the bottom of the picture. In estrogen-dependent LCLs, IG-µ and c-myc expression are mutually regulated by estrogen (B).
Durch das erfindungsgemäße Verfahren können somit zum ersten Mal menschliche Antikörper aus B-Zellen in großen Mengen be reitgestellt werden. Hierzu werden mit EBV oder einem EBV-De rivat immortalisierte B-Zellen verwendet, die das EBNA2-Gen in konditionaler Form enthalten. Zur Immortalisierung der B-Zel len wird das EBNA2-Gen angeschaltet und zur Produktion der Antikörper wird das EBNA2-Gen abgeschaltet.With the method according to the invention, the first Sometimes human antibodies from B cells in large quantities be provided. To do this, use EBV or an EBV-De rivat immortalized B cells used that the EBNA2 gene in conditional form included. For immortalizing the B cell The EBNA2 gene is switched on and used to produce the Antibody switches off the EBNA2 gene.
Die Überstände von Zellkulturen werden bevorzugt zwischen Tag 2 und 5 geerntet und die Antikörper gereinigt. Diese stehen dann zu therapeutischen Zwecken zur Verfügung. Verfahren zur Gewinnung der Antikörper sind aus dem Stand der Technik be kannt und können vom Fachmann auf das vorliegende Verfahren ohne weiteres übertragen werden.The supernatants from cell cultures are preferred between days 2 and 5 harvested and the antibodies cleaned. These stand then available for therapeutic purposes. Procedure for The antibodies are obtained from the prior art knows and can from the expert on the present method can be easily transferred.
Die Antikörper werden vorzugsweise dort verwendet, wo tieri sche oder manipulierte tierische Antikörper zu unerwünschten Nebenwirkungen beim Menschen führen. The antibodies are preferably used where tieri animal antibodies are manipulated or manipulated to produce undesirable Lead to side effects in humans.
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Claims (12)
- a) Immortalisierung menschlicher, Antikörper produzierender B- Zellen mit einem Epstein-Barr-Virus oder einem Deri vat hiervon,
- b) Screening der immortalisierten B-Zellen auf Spezifi tät für das gewünschte Antigen und Isolation von Zellklonen, die entsprechende Antikörper produzieren,
- c) Gewinnung der gewünschten Antikörper aus den Kultur überständen,
- a) immortalization of human, antibody-producing B cells with an Epstein-Barr virus or a derivative thereof,
- b) screening of the immortalized B cells for specificity for the desired antigen and isolation of cell clones which produce corresponding antibodies,
- c) obtaining the desired antibodies from the culture supernatants,
- d) ein Epstein-Barr-Virus oder ein Derivat hiervon mit, in funktioneller Verbindung, einem konditionalen Ep stein-Barr-Virus-Nuclear-Antigen 2-(EBNA2-)Gen oder einem Derivat hiervon verwendet wird, welches zur Immortalisierung zumindest teilweise angeschaltet wird, und
- e) das EBNA2-Gen oder sein Derivat, in funktioneller Verbindung, vor Gewinnung der Antikörper zumindest teilweise abgeschaltet wird.
- d) an Epstein-Barr virus or a derivative thereof is used with, in functional connection, a conditional Epstein-Barr virus nuclear antigen 2 (EBNA2) gene or a derivative thereof which is at least partially switched on for immortalization will, and
- e) the EBNA2 gene or its derivative, in functional connection, is at least partially switched off before the antibodies are obtained.
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19541844A DE19541844C1 (en) | 1995-11-09 | 1995-11-09 | Process for the production of human antibodies and their use |
US08/626,860 US5798230A (en) | 1995-11-09 | 1996-04-03 | Process for the preparation of human monoclonal antibodies and their use |
DE59609000T DE59609000D1 (en) | 1995-11-09 | 1996-10-29 | Process for the production of human monoclonal antibodies and their use |
AT96117361T ATE215563T1 (en) | 1995-11-09 | 1996-10-29 | METHOD FOR PRODUCING HUMAN MONOCLONAL ANTIBODIES AND THE USE THEREOF |
ES96117361T ES2173237T3 (en) | 1995-11-09 | 1996-10-29 | MANUFACTURING PROCEDURE FOR HUMAN MONOCLONAL ANTIBODIES AND THEIR USE. |
EP96117361A EP0773228B1 (en) | 1995-11-09 | 1996-10-29 | Method of producing human monoclonal antibodies and their use |
DK96117361T DK0773228T3 (en) | 1995-11-09 | 1996-10-29 | Process for the preparation of human monoclonal antibodies and their use |
Applications Claiming Priority (1)
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DE19541844A DE19541844C1 (en) | 1995-11-09 | 1995-11-09 | Process for the production of human antibodies and their use |
Publications (1)
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DE19541844C1 true DE19541844C1 (en) | 1997-07-24 |
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Application Number | Title | Priority Date | Filing Date |
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DE19541844A Expired - Fee Related DE19541844C1 (en) | 1995-11-09 | 1995-11-09 | Process for the production of human antibodies and their use |
DE59609000T Expired - Fee Related DE59609000D1 (en) | 1995-11-09 | 1996-10-29 | Process for the production of human monoclonal antibodies and their use |
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DE59609000T Expired - Fee Related DE59609000D1 (en) | 1995-11-09 | 1996-10-29 | Process for the production of human monoclonal antibodies and their use |
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US (1) | US5798230A (en) |
EP (1) | EP0773228B1 (en) |
AT (1) | ATE215563T1 (en) |
DE (2) | DE19541844C1 (en) |
DK (1) | DK0773228T3 (en) |
ES (1) | ES2173237T3 (en) |
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-
1995
- 1995-11-09 DE DE19541844A patent/DE19541844C1/en not_active Expired - Fee Related
-
1996
- 1996-04-03 US US08/626,860 patent/US5798230A/en not_active Expired - Fee Related
- 1996-10-29 EP EP96117361A patent/EP0773228B1/en not_active Expired - Lifetime
- 1996-10-29 ES ES96117361T patent/ES2173237T3/en not_active Expired - Lifetime
- 1996-10-29 DE DE59609000T patent/DE59609000D1/en not_active Expired - Fee Related
- 1996-10-29 DK DK96117361T patent/DK0773228T3/en active
- 1996-10-29 AT AT96117361T patent/ATE215563T1/en not_active IP Right Cessation
Non-Patent Citations (1)
Title |
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EMBO J. 14, S. 88-95, 1995 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19816116A1 (en) * | 1998-04-09 | 1999-10-14 | Gsf Forschungszentrum Umwelt | Preparation of conditionally immortalized immortalization-helper cells, used to immortalize mammalian cells, e.g. for production of hybridomas |
DE19816116C2 (en) * | 1998-04-09 | 2000-06-29 | Gsf Forschungszentrum Umwelt | Process for immortalizing cells using conditionally transformed helper cells |
US6174726B1 (en) | 1998-04-09 | 2001-01-16 | Gsf-Forschungszentrum Fuer Umwelt Und Gesundheit Gmbh | Method for the immortalization of cells using conditionally transformed helper cells |
Also Published As
Publication number | Publication date |
---|---|
ES2173237T3 (en) | 2002-10-16 |
EP0773228A1 (en) | 1997-05-14 |
ATE215563T1 (en) | 2002-04-15 |
DK0773228T3 (en) | 2002-07-22 |
US5798230A (en) | 1998-08-25 |
DE59609000D1 (en) | 2002-05-08 |
EP0773228B1 (en) | 2002-04-03 |
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